Method and device for plankton separation

ABSTRACT

Methods, devices and kits for the physical separation of plankton into its component parts utilizing phototactic behavior are described. The methods utilize positive phototactic behavior and negative contrast orientation of the zooplankton for maximal in situ separation of phytoplankton and zooplankton for use in further studies and evaluation of separation efficiency. The devices provide effective conditions for use in the separation of plankton into component parts.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/936,698, filed on Feb. 6, 2014. The entire teachings of theabove-identified application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Under certain conditions, algae, bacteria and other organisms createhealth hazards for humans and animals through the production of toxinsor bioactive compounds and/or cause deterioration of water quality fromproduction of high biomass. For example, the presence of toxins inrecreational and drinking water can produce many deleterious effects inhumans, including but not limited to fever, headache, muscle and jointpain, blisters, stomach cramps, diarrhea, vomiting, mouth ulcers andallergic reactions. In severe cases, seizures, liver failure andrespiratory arrest may occur. Therefore, increased occurrence of theseorganisms and resultant problems is of great concern.

Improving monitoring techniques for surveillance programs and ecologicalrisk assessments would aid in determining how best to manage theseaquatic ecosystems, thus, helping to ensure that the waterways areproperly managed to maintain their aesthetic, economic, ecological andrecreational value.

SUMMARY OF THE INVENTION

The methods and devices of the invention allow the researcher to collectand separate plankton samples for surveillance programs and ecologicalrisk assessments. Fast, easy and cost effective methods and devices aredescribed herein that overcome existing limitations associated with thecollection, separation and analysis of samples from waterways. Suchlimitations can include, for example, spatial and temporal variability,toxigenicity, and varying sample quality.

Methods, devices (e.g., apparatuses) and kits for separating a planktonsample into its component parts utilizing phototactic behavior aredescribed. Specifically, the methods and devices of the claimedinvention provide the conditions necessary to initiate, direct andreinforce the movement (e.g., migration) of zooplankton away fromphytoplankton in a sample, for use in research requiring separation ofplankton samples, for example, to provide measures of phytoplankton andzooplankton biomass. The separated plankton samples can yield measuresof biomass in different trophic levels. These samples and measurementscan be used for various analyses, including bioaccumulation measurementor evaluation of biological community associations.

In one aspect of the invention, a plankton separating device isdescribed, comprising: a darkened chamber and a collection cartridge(e.g., tube) attached to the chamber for allowing entry of highlydirectional ambient light, wherein the collection tube is of sufficientlength to reinforce migration of the zooplankton, thereby separating theplankton into its component parts.

In one aspect of the invention, a plankton separating device isdescribed, comprising: a darkened chamber having at least one port,wherein the port has a closure; and a collection tube attached to theport of the chamber for allowing entry of highly directional ambientlight, wherein the collection tube is of sufficient length to reinforcemigration of the zooplankton, thereby separating the plankton into itscomponent parts.

In some embodiments, the closure is a stopper or valve. In particularembodiments, the darkened chamber can be configured to be positionedabove the transparent collection tube for operation. The darkenedchamber can have an outer perimeter surrounding a central axis. Thecollection tube can be elongate and extend from the darkened chamberalong the central axis, starting beyond a point that makes about a 48°angle to the central axis while extending to the nearest location ofmaximum outer perimeter dimension of the darkened chamber. This can forma contrast shadow relative to the transparent collection tube,simulating a predator to the zooplankton, minimizing the likelihood thatzooplankton that have migrated into the collection tube will migrateback into the darkened chamber. In this way, the zooplankton migrationinto the tube can be reinforced. Therefore, the collection tube shouldbe of sufficient length to reinforce this negative contrast orientation,and, thus, migration of the zooplankton.

In some embodiments, the collection tube can be transparent and can haveone of tapered or straight side walls. The outer perimeter of thedarkened chamber and the side walls of the transparent collection tubecan be generally round. The transparent collection tube can extend awayfrom the darkened chamber beyond a point that makes about a 20°±2° angleto the central axis while extending to the nearest location of maximumouter perimeter dimension of the darkened chamber. The about 20° anglecan continue to form a contrast shadow relative to the transparentcollection tube that simulates a predator to plankton. At least about40% of the length of the transparent collection tube can extend beyondthe point that makes the about a 20° angle. The darkened chamber and thetransparent collection tube can have outer diameters with a darkenedchamber OD_(b) to transparent collection tube OD_(t) ratio of about3-3.5 to 1. The transparent collection tube can have a length with atransparent collection tube length to OD_(t) ratio of about 3.9-5.2to 1. These ratios can provide a contrast shadow relative to thetransparent collection tube, simulating a predator to zooplankton, andsufficient length in the transparent collection tube for zooplankton tomigrate and move away from the darkened chamber to minimize zooplanktoncollected in the transparent collection tube from migrating back intothe darkened chamber.

The device and methods can utilize ambient light and are able to be usedin situ. In other words, the devices and methods do not require the useof a light source other than ambient light (e.g., bulb, LED or otherillumination). Thus, in some embodiments, the light is ambient. In someembodiments, the device does not include an artificial light source orfilter. In some embodiments the device comprises a reflective surface,such as a mirror or foil. In some embodiments, the level of introducedlight must be of a sufficient level to initiate positive phototacticmovement of the zooplankton to the fluid-filled collection tube. In oneaspect, the change in light intensity is sudden. In some embodiments,the stimulus beam of light can be approximately 2 cm, e.g., 21.5 nm or20 mm.

In some embodiments, the collection tube comprises (e.g., is filledwith) a fluid, preferably water, such as filtered water, e.g., in situfiltered water. In some embodiments, the diluent is in situ filteredwater to maintain thermal and chemical equilibrium of the environmentfor the zooplankton.

In some embodiments, the collection tube is transparent, i.e., entirelytransparent. In another embodiment, most (for example, approximately atleast 85%, e.g., at least 90%, e.g., at least 95%) or all of the tube istransparent, and the remainder of the tube is translucent or opaque. Inanother embodiment, most (for example, approximately at least 85%, e.g.,at least 90%, e.g., at least 95%) or all of the tube is translucent.

In some embodiments, the darkened chamber can have a capacity of atleast about one liter, and the collection tube can have a capacity of atleast about 50 ml. The opening between the darkened chamber and thetransparent collection tube can be, for example, in the range of about19 mm to 22 mm across. The transparent collection tube can have an innerdiameter, with at least a portion of which being about 20 mm to 26 mm.The length of the transparent collection tube can be at least about 110mm. The ratio of the length of dark region (e.g., darkened chamber) tocollection tube length can be about 1-3 to 1.

In one embodiment of the invention, a method for separating plankton isdescribed, comprising acclimating a plankton sample comprisingzooplankton and phytoplankton in a darkened chamber for a sufficientamount of time to facilitate a response to a change in light intensity;introducing light at a sufficient level to initiate phototactic movementto a collection tube filled with a fluid (e.g., water, for example,filtered water, such as in situ filtered water), wherein the zooplanktonis separated from the phytoplankton; collecting a zooplankton samplefrom the device; and collecting a phytoplankton sample from the device,wherein the plankton is separated to zooplankton and phytoplankton. Inparticular embodiments, the plankton sample can be a concentratedsample. The concentrated sample can be diluted.

In another embodiment of the invention, a method for separating planktonis described, comprising acclimating a plankton sample comprisingzooplankton and phytoplankton in a darkened chamber for a sufficientamount of time to facilitate a response to a change in light intensity;introducing light at a sufficient level to initiate positive phototacticmovement of the zooplankton to a fluid-filled collection tube, said tubebeing of sufficient length to reinforce negative contrast orientation,wherein zooplankton is separated from phytoplankton, collecting azooplankton sample from the collection tube; and collecting aphytoplankton sample from the collection tube, wherein the plankton isseparated to zooplankton and phytoplankton. In one embodiment of thefirst aspect, the method further includes analyzing (e.g., studying) thesample. In some embodiments, analysis can comprise, e.g.,identification, enumeration, and/or quantification of biomass,quantification of pigment fluorescence, etc.

In some embodiments, the invention relates to a method for separatingplankton, comprising placing a plankton sample comprising zooplanktonand phytoplankton in a darkened chamber; acclimating the plankton for asufficient amount of time to facilitate a response by the zooplankton toa change in light intensity; and introducing ambient light to thechamber to initiate phototactic movement of the zooplankton to acollection tube filled with water, the phototactic movement into thecollection tube separating the zooplankton from the phytoplankton. Insome embodiments, the collection tube is of sufficient length toreinforce contrast orientation. In some embodiments, collection tube istransparent. In some embodiments, the collection tube is located belowthe darkened chamber at a 90° angle relative to a horizontal base of thedarkened chamber. In some embodiments, the collection tube has a lengthsufficient to ensure that an angle of 48° to normal can be achieved bythe zooplankton. In some embodiments, the plankton is acclimated for 20minutes or less.

In some embodiments, the invention relates to a plankton separationmethod comprising introducing a plankton sample comprising zooplanktonand phytoplankton to a darkened chamber of the devices described herein,acclimating the sample for a sufficient amount of time to facilitate aresponse to a sudden change in light intensity; introducing highlydirectional ambient light at a sufficient level to initiate phototacticmovement to a collection tube filled with water, said tube of sufficientlength to reinforce negative contrast orientation, wherein thezooplankton is separated from the phytoplankton, collecting zooplanktonfrom the collection tube; and collecting phytoplankton from thecollection tube, wherein the plankton is separated to zooplankton andphytoplankton samples.

In some embodiments, the invention relates to a plankton separatingdevice comprising a darkened chamber having a port, wherein the port hasa closure; and a collection tube attached to the port of the chamber forallowing highly directional ambient light, wherein the collection tubeis of sufficient length to reinforce migration of the zooplankton,thereby separating plankton into its component parts, wherein theclosure is configured to be changed from a closed state to an open statewith the collection tube attached to the port. In some embodiments, theclosure is a stopper or valve. In some embodiments, the chamber isconfigured to be positioned above the collection tube during operation,the chamber having an outer perimeter surrounding a central axis, thecollection tube being elongated and extending from the darkened chamberalong the central axis, starting beyond a point that makes about a 48°angle to the central axis while extending to the nearest location ofmaximum outer perimeter dimension of the darkened chamber. In someembodiments, the outer perimeter of the darkened chamber and the sidewalls of the collection tube are generally round.

In some embodiments of the methods, devices and kits described herein,the collection cartridge (tube) is of sufficient length to reinforcecontrast orientation. In some embodiments, collection tube istransparent. In some embodiments, the collection tube is located belowthe darkened chamber at a 90° angle relative to a horizontal base of thedarkened chamber. In some embodiments, the collection tube has a lengthsufficient to ensure that an angle of 48° to normal can be achieved bythe zooplankton. In some embodiments, the collection tube is of asufficient length to reinforce migration. In some embodiments, thecollection tube has one of tapered or straight side walls.

In some embodiments, the collection tube extends away from the darkenedchamber beyond a point that makes about a 20°±2° angle to the centralaxis while extending to the nearest location of maximum outer perimeterdimension of the darkened chamber. In some embodiments, at least about40% of length of the collection tube extends beyond said point thatmakes said about a 20° angle. In some embodiments, the darkened chamberand the collection tube have outer diameters with a darkened chamber ODbto transparent collection tube ODt ratio of about 3-3.5 to 1, thetransparent collection tube having a length with a transparentcollection tube length to ODt ratio of about 3.9-5.2 to 1, therebyproviding a contrast shadow relative to the transparent collection tubesimulating a predator to plankton, and sufficient length in thetransparent collection tube for plankton to migrate from and move awayfrom the darkened chamber to minimize plankton collected in thetransparent collection tube from migrating back into the darkenedchamber. In some embodiments, the darkened chamber has a capacity of atleast about one liter, and the collection tube has a capacity of atleast about 50 ml. In some embodiments, the opening between the darkenedchamber and the collection tube is in the range of about 19 to about 22mm across. In some embodiments, the collection tube has an innerdiameter, at least a portion of which being about 20 mm to about 26 mm.In some embodiments, the length of the collection tube is at least about110 mm.

In another aspect, a plankton separation method is described using adevice of the invention, the method comprising acclimation of a planktonsample comprising zooplankton and phytoplankton in a darkened chamber ofthe device for a sufficient amount of time to facilitate a response to achange (e.g., a sudden change) in light intensity; introducing highlydirectional ambient light for phototactic movement of zooplankton fromthe darkened chamber to a fluid-filled collection tube of sufficientlength to reinforce negative contrast orientation; collectingzooplankton from the collection tube; and collecting phytoplankton fromthe collection tube, wherein the plankton is separated to zooplanktonand phytoplankton.

In another aspect, kits are described. In one embodiment, a kitcomprising a plankton separation device of the invention andinstructions for using the device is described. In another embodiment,the kit can comprise a darkened chamber and a collection tube. Inanother embodiment, the kit further comprises filtered water.Educational materials can be included in with kits. Educationalmaterials can include, but are not limited to, any materials which serveto impart knowledge, information, or skills, including, but not limitedto, instructions for how to use the device; information regarding how toanalyze the samples; information regarding water quality, water studies,and/or plankton; and suggestions for age and/or ability appropriateactivities and lab exercises, including for those in age group K-12.

In another aspect, the invention encompasses methods of measurement andassays of plankton and plankton related materials using the methods anddevices described herein. For example, in one embodiment, themeasurement is a measurement of planktonic biomass.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingembodiments of the present invention.

FIGS. 1A-1C show an embodiment of the separation device, having adarkened chamber (10-1; amber bottle), closure for temporary physicalseparation (20-1, stopper), collection tube (30-1; tube with taperedend) and optional external support (40-1; sling support).

FIGS. 1D-1F show other embodiments of the device, with external supportof rings, a table support, and a c-clamp system.

FIGS. 1G-1I shows further embodiments of the separation device.

FIG. 1J is a front view of the embodiment of FIG. 1A with annotations.

FIG. 1K is a front view of the embodiment of FIG. 1D with annotations.

FIG. 1L is a front view of a ball valve connected to a filter cone andcartridge.

FIG. 1M is a side view of an embodiment of a separation device in thepresent invention including a securement arrangement.

FIG. 1N is a side view showing another configuration of the securementarrangement of FIG. 1M.

FIG. 2 is a plot showing the separation efficiency for macrozooplankton(top line) and microzooplankton (bottom line) on Aug. 1, 2013(Experiment 1) “Z”=zooplankton, 50 mls., “P”=phytoplankton, 900 mls.

FIG. 3A and FIG. 3B are plots showing mean separation efficiencies formacrozooplankton and microzooplankton in Lake Cochichewick (A) andWilland Pond (B) on Sep. 4 and Sep. 5, 2013, respectively.

FIG. 4A and FIG. 4B are plots showing calibration curves formacrozooplankton (solid line), cyanobacteria (dashed line), and allphytoplankton (dotted line) for Lake Cochichewick (A) and Willand Pond(B) on Sep. 4 and Sep. 5, 2013, respectively. Confidence intervals (95%)shown as gray lines. Df=4. “Z”=zooplankton, 50 mls, “P”=phytoplankton,900 mls.

FIG. 5A and FIG. 5B are plots showing calibration curves formacrozooplankton (solid line) and cyanobacteria (dashed line) for LakeCochichewick (A) and Willand Pond (B) compared with data from Oct. 10and Oct. 16, 2013, respectively.

FIG. 6 is a graph showing separation efficiency curves for LakeCochichewick on Aug. 1, 2013. Macrozooplankton (solid line) andmicrozooplankton (solid gray line). Z″=50 mls, “P”=950 mls.

FIGS. 7A and 7B are graphs showing separation efficiency curves formacrozooplankton biomass versus microcystis equivalents and chlorophyllin Lake Cochichewick on Sep. 4, 2013 and Oct. 10, 2013.

FIGS. 7C and 7D are graphs showing separation efficiency curves formacrozooplankton biomass versus microcystis equivalents and chlorophyllin Willand Pond Sep. 5, 2013 and Oct. 16, 2013.

FIGS. 8A and 8B are graphs showing separation efficiency curves formacrozooplankton biomass versus microcystis equivalents and chlorophyllin Lake Cochichewick Sep. 4, 2013, Oct. 10, 2013, and Oct. 29, 2014.

FIGS. 8C and 8D are graphs showing separation efficiency curves formacrozooplankton biomass versus microcystis equivalents and chlorophyllin Willand Pond Sep. 5, 2013, Oct. 16, 2013 and Sep. 6, 2014.

FIG. 9 is a graph depicting effect of minimum adapter diameter onseparation efficiency. Macrozooplankton in Lake Cochichewick 29 Oct.2014 with standard errors for each shown. Ambient (t-3.54, df-4,p-0.024), Artificial (t-4.90, df-4, p-0.008).

FIG. 10 are graphs showing separation efficiencies for individualzooplankters from Lake Cochichewick on Oct. 29, 2014 and Willand Pond onSep. 6, 2014.

FIG. 11 is a table of separation efficiencies for zooplankton biomass asobserved in Lake Cochichewick 2013-2014.

FIG. 12 is a table of separation efficiencies for zooplankton biomass asobserved in Willand Pond 2013-2014.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

Described herein are methods and devices for limnological studies usingplankton separation, for analysis, bioaccumulation selectivity andevaluation of biological community associations. It was found that themethods and devices of the invention allow the researcher to rapidlycollect samples for improved routine surveillance programs andecological risk assessments in situ.

The structure of planktonic populations in the aquatic ecosystems isdynamic and constantly changing in species composition and biomassdistribution. Changes in species composition and biomass distributionmay affect separation efficiency.

Plankton, particularly phytoplankton, have long been used as anindicator of water quality. Because of their short life spans, planktonresponds quickly to environmental changes. Some species are verysensitive to organic and/or chemical wastes. Some species have also beenassociated with noxious blooms causing toxic conditions apart from tasteand odor problems. The presence of toxins and potential forbioaccumulation threaten fresh water ecosystems, humans and animals.

The physical and chemical characteristics of water affect the abundance,species composition, stability and productivity of indigenouspopulations of aquatic organisms. The biological methods used foranalyzing (e.g., assessing) water quality include, but are not limitedto, collection, counting and identification of aquatic organisms;biomass measurements; measurements of metabolic activity rates; toxicitytests; potential for bioaccumulation of pollutants; and processing andinterpretation of biological data. The work involving plankton analysisaids in the explanation of the cause of color and turbidity and thepresence of objectionable odor, tastes and visible particles in waters;the interpretation of chemical analyse; and the identification of thenature, extent and biological effects of pollution. It also providesdata on the status of an aquatic system on a regular basis. The processof plankton separation provides a sample of adequate size and improvedquality for postanalytical techniques that include, but are not limitedto, assays such as enzyme-linked immunosorbent assay (ELISA), inhibitionassays and radioassays.

Numerous studies have been conducted on the occurrence of thecyanobacteria and the toxins that they produce (Carmichael and Falconer1993, Yoo et al., 1995, Carmichael 1997). Many studies have beenconducted to further our understanding of the complex dynamics ofcyanobacterial abundance and community composition as they are affectedby water temperature, solar irradiance, hydrology, nutrient supply andmeteorological conditions. A report (Lopez et al. 2008) outlines currentand future efforts that would support and expand understanding of thecyanobacteria, cyanotoxins, ecological impacts, human health effects andmanagement techniques. In the report, the need to improve monitoringtechniques was recommended for surveillance programs and ecological riskassessments. For example, routine surveillance programs can be improvedwith the use of the cost-effective methods and devices described hereinto determine the relative contribution of the cyanobacteria to thephytoplankton assemblage. Additionally, the toxigenicity of thecyanobacterial community can be assessed with a rapid, cost effectivemethod to obtain samples that yield precise measures of phytoplanktonbiomass and weight specific toxicity. This information can be used todetermine trends in the ecological integrity of the aquatic systems andsupport the decision making process regarding use attainment. Ecologicalrisk assessments of bioaccumulation in zooplankton (i.e., bulkzooplankton, macrozooplankton) can be simplified and improved with arapid, cost effective method to obtain samples that yield precisemeasures of zooplankton biomass and weight specific toxicity.

Sampling and monitoring of waterways is largely done by state andfederal agencies with assistance from volunteers. Methods and devicesthat are easy to use and do not require expensive or complex systems orparts for obtaining samples are needed. The methods and devices of thepresent invention meet these needs. Routine surveillance programs arebenefited by the cost effective methods and devices described hereinthat can assess the toxigenicity of the cyanobacterial community. Thisinformation can be used to determine trends in ecological integrity andsupport the decision making process regarding use attainment. Thecalculation of dry weight biomass and weight specific toxicity issimplified and improved with the methods of the invention for separatingplankton samples into its component parts. Routine surveillance programsusing biological community associations can be enhanced with a rapidmethod for collection of samples for analysis. Furthermore, ecologicalrisk assessments are improved using the methods and devices for theevaluation of toxin in different trophic levels, includingquantification of cyanotoxins in phytoplankton and the resultingaccumulation in zooplankton.

Previous studies have utilized phototactic behavior to separate planktoninto its components to quantify cyanotoxins in the phytoplankton andsubsequent transfer to the zooplankton (Capron 1995, Johnson 1999,Hathaway 2001, Larsson et al. 2001, Jonasson et al., 2010, Haney 2013,Jonasson, 2013). Phototactic behavior (swimming) is a stimulus responsethat requires a velocity (kinesis) and a direction (orientation). Totake advantage of this naturally occurring phenomenon, the researchermust establish a set of necessary conditions before the phenomenonoccurs (Nagel 1974). A hierarchy of response (Loose 1993) to stimuluswould include the relative change in light intensity (Ringelberg 1964)which would exceed the rheobase (Ringelberg, 1964, Daan and Ringelberg,1969) necessary to initiate a swimming response. A positive phototacticresponse could be anticipated as a result of exposure to a narrowstimulus beam (Forward 1988) (highly directional light) with an angularlight distribution that approximates 0° (Schallek 1942). Body axisorientation would result from dorsal beam contrast (45° or less)(Ringelberg 1964) (Ringelberg, Flik and Buis 1975) that would controlthe direction of movement in the vertical plane. The orientation of thedevice (darkened above, light below) serves to reinforce body axisorientation as a flight response from predators. The swimming velocity(Daan and Ringelberg, 1969) would have to be sufficient to migrate thedistance in the time allowed. Any barriers such as spatial requirements,temperature, pressure, angular light distribution, and otherenvironmental conditions would have to be overcome. In previous studiesas noted, the necessary conditions for the phenomenon to occur were metwith each researcher modifying the conditions somewhat (spatialrequirements, light source, time, distance and temperature). Forexample, previous researchers provided illumination, followed by waiting2 hours, five (5) minutes, 15 minutes and twenty (20) minutes beforecollecting their respective zooplankton samples. In addition, variousvolumes were collected. These methods resulted in reduced separationefficiency.

The methods and devices of the invention allow for the qualitative andquantitative analysis of plankton. Such studies can monitor the impactof environmental changes on ecological integrity. The methodology anddevices simplify and reduce costs associated with monitoring programswhile improving the accuracy of the data collected. The device andmethods described herein utilize phototactic behavior and contrastorientation for maximal in situ separation of phytoplankton andzooplankton. Further, gathering quantitative data on separationefficiencies, the development of conditions necessary for a desiredresult based on research objectives can be achieved.

As used herein, “plankton” refers to a diverse group of organisms thatlive in fresh or salt water. Plankton is usually free floating,suspended in water, nonmotile or insufficiently motile to overcometransport by water currents. Plankton includes phytoplankton andzooplankton.

Phytoplankton generally live near the water surface where there issufficient light to support photosynthesis. Examples of phytoplanktoninclude, for example, algae, diatoms, cyanobacteria, dinoflagellates andcoccolithophores. Phytoplankton can be, for example, unicellular,colonial or filamentous, and is autotrophic (primarily photosynthetic)and can be eaten by zooplankton and other organisms occurring in thesame environment.

Cyanobacteria is photosynthetic bacteria found in freshwater and marineenvironments, including lakes, streams, ponds, the ocean and othersurface waters. Cyanobacteria can include planktonic cells orphototrophic biofilms. It can reproduce exponentially to form extensiveand highly visible blooms. This blooming cyanobacteria can producecyanotoxins in such concentrations that they poison and even killanimals and humans. Cyanotoxins can also accumulate in other animalssuch as fish and shellfish, and cause poisonings such as shellfishpoisoning. Among cyanotoxins are some of the most powerful naturalpoisons known, including poisons which can cause death by respiratoryfailure. The toxins include neurotoxins, cytotoxins, hepatotoxins, andendotoxins.

Zooplankton include, for example, microscopic protozoans, rotifers,cladocerans and copepods and other aquatic organisms. The speciesassemblage of zooplankton also may be useful in assessing water quality.Zooplankton can be further separated into size classes, such asmacrozooplankton and microzooplankton. Macrozooplankton include, but arenot limited to, microcrustaceans larger than 63 ums (microns), includingbut not limited to Cladocerans: Bosmina spp., Chydorineae spp.,Ceriodaphnia spp., Daphnia spp., Diaphanosoma spp.; and Copepods:Calanoids-female, Calanoids-male (Diaptomus spp.), Microcyclops spp.,Mesocyclops spp., and all stages of copepodites. Microzooplanktoninclude, but are not limited to, microcrustacean nauplii and rotiferslarger than 20 um, such as 20-63 microns, including, but not limited to:Keratella spp., Kellicottia spp., Trichocera spp., Asplancha spp., andAscomorpha spp.

As used herein, “phototaxis” refers to locomotory movement that occurswhen a whole organism moves responds to a relative change in lightintensity. This can be advantageous for phototrophic organisms as theycan orient themselves most efficiently to receive light forphotosynthesis. Phototaxis is positive if the movement is in thedirection of increasing light intensity and negative if the direction isopposite. The variables that initiate phototactic behavior and maximizemigration in the present disclosure include but are not limited to, therelative change in light intensity (e.g., without the use of anartificial light source or filter(s)).

As used herein, “contrast orientation” refers to locomotory movementsthat occur when a whole organism responds to a spatial change in lightintensity. This is advantageous to phototactic organisms as they canorient themselves most efficiently to respond to light/dark boundariesthat may indicate the presence or absence of predators. Contrastorientation can be positive or negative.

In some embodiments, the plankton is acclimated in the chamber.Acclimation for a “sufficient amount of time” means a sufficient amountof time to facilitate a response by the plankton to a change in lightintensity, for example, between about 20 and about 45 minutes. In oneembodiment, the time is about 20 minutes or less, e.g., about 20minutes.

The selection of plankton separation times can be based on a number offactors, including, for example, the potential for reverse migration bythe zooplankton (for example, about 0-60 minutes, e.g., 1 hr.) andphytoplankton contamination of the zooplankton portion as a result ofgravity. The desirable phytoplankton “contamination” level typicallydoes not exceed 5%.

As used herein, “sampling” refers to collecting a sample, e.g., a watersample, comprising plankton for monitoring. As used herein, “migrationpotential” is the distance traveled by an organism in a desiredtimeframe.

As used herein, “plankton net” refers to a type of field equipment usedto trap plankton. It typically has a polyethylene filter of a definedmesh size and a graduated measuring jar attached to the other end. Ahandle or ring can hold the net. The mesh size of the net determines thesize range of the plankton trapped. For example, a mesh of 50 ums can beused for collecting samples.

Example devices are shown in FIGS. 1A-1I. The chamber (10-1) can be of adark color (e.g., black, amber) and constructed of a durable material,in some embodiments with a conical shape and smooth walled. The chamberis constructed so that light is prevented from entering the chamberduring the separation phase. For example, the chamber can have at leastone port (12) with a closure for temporary separation (20) (e.g.,stopper with plastic rod (20-1), valve, ball valve (20-2) screw cap, orother mechanism to stop fluid communication as needed). In anotheraspect, the chamber has one or more additional sample port(s) (14) forintroduction of the water and/or sample. In certain embodiments, thesample port is located at the top of the chamber and can be of asufficient size for introduction of sample. The sample port (opening)includes a closure (16), for example a cap (16-1). The port (14) can actas a sample port if there is only one port. The port can have openingsto the interior of the chamber that further have a closure.

During the separation phase, the internal chamber is darkened and atemporary physical separation (e.g., a closure) between the chamber anda collection tube (30) is removed. The collection tube is of asufficient size and material to facilitate migration of the zooplankton.The collection tube is attached to the chamber (10-1) via a port (12)and is transparent or translucent or can become transparent for allowinga sufficient amount of highly directional ambient light to enter andfacilitate collection of zooplankton.

In a certain embodiment, the collection tube is conical in shape tofacilitate the collection of zooplankton. The length of the collectiontube can be selected so that the zooplankton can migrate past a 48°angle to the normal within the tube. The collection tube optionallyincludes a valve (32) e.g., a ratchet valve (32-1), or clamp to permitor stop flow of the zooplankton. The collection tube can be a taperedtube. In alternative embodiments, the tube has a screw cap on one endand blunt end with cap on the other. The collection tube is ofsufficient length for migration and separation of the components of theplankton. In certain embodiments, the separation chamber includes anexternal support to the device (40) for example, a bridle/sling (40-1)assembly and/or external rings, for positioning the chamber for use witha table stand (40-2) or a clamping device (40-3).

Referring to FIG. 1J, the darkened container or chamber (10-1) ofseparating device 6 for containing a sample 11 of water and plankton forseparation, can be a generally cylindrical bottle with a circular orround outer perimeter or side wall, and have a height H with a generallyconstant outer diameter OD_(b) that concentrically surrounds alongitudinal central axis X. The collection chamber, region, containeror tube (30-1) for containing filtered water 13 into which desiredplankton 15 can be collected, can be connected to and sealed to thedarkened chamber (10-1) at a central outlet, opening or port (12). Thecollection tube (30-1) can extend in a straight manner from the darkenedchamber (10-1) along or aligned with the longitudinal central axis X.The collection tube (30-1) is typically transparent and typicallyoperates with existing ambient light, and can have a tapered shape orside walls, being widest at port (12) and narrowest at ratchet clamp orvalve (32-1). In some embodiments, the device comprises a single port.

The separating device 6 can be configured to be used in operation withthe darkened chamber (10-1) being positioned above the transparentcollection tube (30-1), for example with the longitudinal central axis Xbeing vertical or near vertical. This can allow plankton such asphototactic zooplankton that are attracted to light, such as ambientlight on, within or illuminating the transparent collection tube (30-1),to move or swim vertically downwardly from darkened chamber (10-1) withgravity into the transparent collection tube (30-1). Higher percentagesof such plankton tend to swim vertically downwardly with gravity tolight, in comparison to swimming to light horizontally or laterally, orvertically upwardly against gravity. Therefore, positioning thetransparent collection tube (30-1) vertically below the darkened chamber(10-1) can maximize desired plankton migration toward light to obtainmaximum or high separation efficiencies. In addition, the port (12)between the darkened chamber (10-1) and the transparent collection tube(30-1) can have a small opening in comparison to the outer perimeterdiameter OD_(b) (about ⅓ in size), which produces a narrow definedcircular beam or spot of light with high contrast from the transparentcollection tube (30-1) vertically upwardly from below along longitudinalaxis X into darkened chamber (10-1), which draws phototactic plankton 15downwardly vertically into the transparent collection tube (30-1). Ifthe port (12) is too large, too much light can diffuse into the bottomof the darkened chamber (10-1), and not provide enough contrast ordefinition between dark and light to cause the plankton 15 to migrateinto the transparent collection tube (30-1).

The 48° to normal cone angle, is the angle A which is measured 48°relative to the longitudinal central axis X and a line extending from apoint 38 along longitudinal axis X that intersects or passes through ahorizontal or lateral base line 42 at the widest or maximum perimeter ordiameter side wall dimension location of darkened chamber (10-1) that isclosest to the transparent collection tube (30-1). The longitudinal axisX is normal to lateral line 42. A shadow of an object such as darkenedchamber (10-1) above plankton 15 (such as zooplankton that have migratedinto transparent collection tube (30-1)), at a cone angle of 48° orless, can form a concentric contrast shadow relative to the plankton 15within the interior of the transparent collection tube (30-1). Thatcontrast shadow can simulate a predator to the plankton 15, which tendsto cause the plankton 15 to swim downwardly within the transparentcollection tube (30-1) away from the darkened chamber (10-1) to maintainseparation of the plankton in separating device 6. If angle A is largerthan 48°, the shadow of the darkened chamber (10-1) typically does notprovide enough contrast between light and dark to the plankton 15 tosimulate a predator, and some of the plankton 15 within the transparentcollection tube (30-1) tends to migrate back into the darkened chamber(10-1). As can be seen in FIG. 1J, the 48° angle A is located within thedarkened chamber (10-1), and the cone angle B measured relative tolongitudinal axis X and a line extending from a point 44 alonglongitudinal axis X, at the transition between the darkened chamber(10-1) and the transparent collection tube (30-1), to the outerperimeter of darkened chamber (10-1) on base line 42, is less than 48°.Angle B is the angle that plankton 15 can migrate past and view theconcentric contrast shadow of the darkened chamber (10-1). This angle Bis less than 48°, such as 41° in some embodiments, and forms aconcentric angle and contrast shadow simulating a predator in alldirections when the darkened chamber (10-1) and the transparentcollection tube (30-1) are both round, that drives the plankton withinthe transparent collection tube (30-1) downwardly away from the darkenedchamber (10-1). The transparent collection tube (30-1) is sufficientlylong enough to allow the plankton 15 to swim far enough downwardly awayfrom the darkened chamber (10-1) with gravity to not migrate upwardlyback into the darkened chamber (10-1) against gravity.

In some embodiments, the darkened chamber (10-1) can be a lightimpermeable plastic, glass or metal bottle for holding a sample 11 ofabout 1 liter, and the transparent collection tube (30-1) can be a clearor transparent tapered elongate plastic or glass tube for holding orcontaining about 50 ml of filtered water 13 and collected plankton 15.The darkened chamber (10-1) can have a height H of about 166 mm (6.5 in)with a maximum outer perimeter diameter OD_(b) portion of about 94 mm(3.7 in) that is constant until reaching the top of the darkened chamber(10-1). The darkened chamber (10-1) can narrow down from the outerperimeter diameter OD_(b) of 94 mm at line 42 to about 30 mm (1.2 in) atport (12) over a distance Y that can be about 50 mm (2 in). Thetransparent collection tube (30-1) can have a length L of about 150 mm(5.9 in) between port (12) and ratchet valve (32-1). The transparentcollection tube (30-1) can be round and have a maximum outer diameterOD_(t) at port (12) of about 29.5 mm (1.2 in) with a corresponding innerdiameter 1D_(t) of about 21.5 mm (0.85 in). The opening into thetransparent collection tube (30-1) from darkened chamber (10-1) can beabout 21 mm±2 mm (0.83 in ±0.08 in). It has been found that smalleropenings into the transparent collection tube (30-1), such as 13 mm,hinder the migration of phototactic plankton and result in lowerseparation efficiencies. At the ratchet valve (32-1) at the bottom, theouter diameter can taper to about 10 mm (0.4 in) with a correspondinginner diameter of about 7 mm (0.3 in). Port (14) at the top of darkenedchamber (10-1) can have a diameter of about 69 mm (2.7 in). Angle B canbe about 41°±2°. The transparent collection tube (30-1) can form anarrow circular or round tapering column of water exposed to ambientlight, extending downwardly concentrically from darkened chamber (10-1),starting with about ⅓ the diameter of the darkened chamber (10-1).

A cone angle C of about 20°±2°, such as 19°, can extend relative to thelongitudinal axis X and a line extending from point 40 alonglongitudinal axis X within transparent collection tube (30-1) to themaximum outer perimeter diameter OD_(b) at base line 42. About 40% ofthe length of transparent collection tube (30-1) can extend downwardlybelow the 19° angle C. This provides enough downwardly vertical spacewithin transparent collection tube (30-1) where collected plankton 15can swim downwardly far enough away from darkened chamber (10-1) inresponse to the simulated predatory contrast shadow produced, where theplankton will not migrate back into the darkened chamber (10-1). Thedarkened chamber outer diameter OD_(b) and the transparent collectiontube outer diameter OD_(t) can have an OD_(b) to OD_(t) ratio of about3-3.3 to 1, such as about 3.2 to 1, and there can also be an OD_(b) totransparent collection tube inner diameter ID_(t) ratio, OD_(b) to1D_(t) ratio of about 4.2-4.6 to 1, such as about 4.4 to 1. Thetransparent collection tube (30-1) can have a length L to outer diameterOD_(t) ratio of about 5-5.2 to 1, such as 5.1 to 1, and a L to 1D_(t)ratio of about 6.8-7.2 to 1, such as about 6.9 to 1. The ratio of thedark region length of darkened chamber (10-1) to transparent collectiontube length L can be about 1.2-1.6 to 1 such as about 1.4 to 1. Suchconfigurations, dimensions and ratios can maximize separationefficiencies of plankton separation, by using plankton's migrationresponses to light and predators.

Referring to FIG. 1K, the darkened chamber (10-1) of separating device 8can be similar to that in separating device 6, and can have similarconstruction, shape and dimensions as previously discussed. Separatingdevice 8 can differ in that instead of having a closure stopper (20-1)for initially separating the sample 11 within the darkened chamber(10-1) from the transparent collection tube (30-1), a valve (20-2) suchas a ball valve, can be mounted or connected to port (12) of thedarkened chamber (10-1), and a transparent collection chamber, region,container or tube (30-2) for typically operating in ambient light, canbe connected to the bottom or lower end or outlet of valve (20-2). Thelocation of base line 42 and the 48° cone angle A relative to darkenedchamber (10-1) are similar to that in separating device 6. However, thevalve (20-2), which can be dark or light impermeable, forms a longerdarkened region relative to base line 42 along longitudinal axis X,before reaching transparent collection tube (30-2), that can have adistance Z of about 135 mm (5.3 in). The valve (20-2) can have anopening therethrough with an inner diameter of about 20 mm (0.78 in)±2mm (0.08 in). The valve (20-2) connected to the darkened chamber (10-1)can form a narrow circular dark column extending concentrically downwardfrom darkened chamber (10-1) before reaching transparent collection tube(30-2), that can be about 121 mm (4.8 in) long. The transparentcollection tube (30-2) can extend in a straight manner from valve (20-2)along longitudinal axis X, a length of about 111 mm (4.4 in), and can beround or cylindrical with a side wall having a constant outer diameterOD_(t) of about 28 mm (1.1 in) and an inner diameter 1D_(t) of about 25mm (0.98 in). The collection tube (30-2) can form narrow circular, roundor cylindrical column of water 13 exposed to ambient light, extendingdownwardly concentrically from darkened chamber (10-1) and valve (20-2),having about ⅓ the diameter of darkened chamber (10-1).

The transparent collection tube (30-2) can be used for containing about50 ml of filtered water 13 and collected plankton 15. The use of theball valve (20-2) instead of closure stopper (20-1) can provideseparating device 8 with more consistent separation results than withseparating device 6. The ball valve (20-2) can open the path or port(12) between the darkened chamber (10-1) and the transparent collectiontube (30-2) in a repetitive smooth consistent manner, with a twist of aknob. With regard to closure stopper (20-1) in separating device 6, astopper is pushed into or pulled from port (12) by a stick or rod. Coneangle B is measured relative to longitudinal axis X and a line extendingfrom a point 40 along longitudinal axis X at the transition between thedarkened valve (20-2) and the transparent collection tube (30-2), thatintersects the outer diameter OD_(b) at base line 42, and is less than48°. In some embodiments, angle B can be about 20°±2°, such as 19° andcan form a concentric contrast shadow of the darkened chamber (10-1)relative to the plankton 15 within the interior of the transparentcollection tube (30-2) that effectively simulates a predator to theplankton 15. This can cause the plankton 15 to swim downwardly withgravity within transparent collection tube (30-2) away from the darkenedchamber (10-1) to maintain separation of desired plankton. Although a19° angle B is less than half that of 48°, the 19° angle is veryeffective to prevent plankton 15 within transparent collection tube(30-1) from migrating back into darkened chamber (10-1) against gravity,and the full length L of transparent collection tube (30-2) extendsbelow point 40 of the 19° angle B to allow plenty of room for theplankton 15 to migrate downwardly away from darkened chamber (10-1) andvalve (20-2) with gravity. The darkened chamber outer diameter OD_(b)and the transparent collection tube outer diameter OD_(t) can have anOD_(b) to OD_(t) ratio of about 3.3-3.5 to 1, such as 3.4 to 1, andthere can also be an OD_(b) to transparent collection tube innerdiameter ID_(t) ratio, OD_(b) to ID_(t) ratio of about 3.5-4 to 1, suchas 3.8 to 1. The transparent collection tube (30-2) can have a length Lto outer diameter OD_(t) ratio of about 3.9-4.2 to 1, such as 4 to 1,and a L to 1D_(t) ratio of about 4.2 to 4.6 to 1, such as 4.4 to 1. Theratio of the dark region length consisting of darkened chamber (10-1)and valve (20-2) to transparent collection tube length L can be about2.5-2.9 to 1, such as 2.7 to 1. About 40% of the darkened region can bea narrow or circular column extending through valve (20-2). Theseconfigurations, dimensions and ratios can also maximize separationefficiencies of plankton separation, and also uses plankton's migrationresponses to light and predators. Although separating devices 6 and 8typically make use of ambient light entering transparent collectiontubes (30-1) and (30-2). If desired, a reflector or a light source 46can be used and positioned below or to the side of the devices 6 and 8for directing light 46 a upwardly into collection tubes (30-1) and(30-2).

The separation devices 6 and 8 are able to obtain higher separationefficiencies of plankton than prior devices. The stopper (20-1) or valve(20-2) can keep the sample 11 to be separated, both physically andphototactically isolated within the darkened chamber (10-1) from thetransparent collection tubes (30-1) and (30-2), until opened. Thevertical orientation of the darkened chamber (10-1) being above thetransparent collection tubes (30-1) and (30-2) with a circular beam orspot of light being directed vertically upward through a relativelysmall port (12) in the darkened chamber (10-1) provides defined light todark contrast that promotes initial migration of phototactic plankton 15downwardly toward the light while assisted by and in the direction ofgravity. The opening between the darkened chamber (10-1) and thetransparent collection tube is sized small enough to provide anattractive defined high contrast beam of light, while large enough notto impede plankton migration. An opening that is too large can let toomuch light into the darkened chamber (10-1) so there is not a sufficientlight to dark contrast, and not promote migration. The transparentcollection tubes extend straight down from the darkened chamber (10-1)so that migrating plankton 15 can swim past a 48° angle or less to thelongitudinal axis X as described above, such as beyond a 41° point or a19° or 20° point, within the transparent collection tube. The plankton15 view the concentric contrast shadow of the darkened chamber (10-1) asa predator and tend to move downwardly with gravity, and not migrateback into the darkened chamber (10-1) against gravity. By having a largeenough collection sample, such as at least 50 mls, the transparentcollection tube can have a length that is long enough for the plankton15 to swim far enough downwardly away from the darkened chamber (10-1)and not to migrate back in. The transparent collection tube can extendat least about 40% of the length L of the transparent collection tubebeyond the point that makes about a 20°±2° angle to the longitudinalaxis X as previously described. In separating device 8, the whole lengthL of transparent collection tube (30-2) extends beyond the point of the19° or 20° angle. By having a relatively narrow diameter transparentcollection tube, the contrast shadow that the plankton 15 therein sees,can have a relatively consistent viewing angle in all directions.Although particular dimensions have been given, the dimensions can vary,for example, larger darkened chambers and transparent collection tubescan be used.

Referring to FIG. 1L, valve (20-2) can be connected to a phytoplanktoncartridge and filter cones for processing, as desired.

Referring to FIG. 1M, separating device 8 can be mounted by a mountingdevice bracket or arrangement 48, for example, to a surface or rail,such as on a boat or canoe for use or testing on the water. The mountingdevice 48 can have a C-clamp 26 for securement to the desired surface orrail 24. A bracket body 28 can have a portion 28 a secured to or arounda vertical member of the C-clamp 26, and a portion 28 b for rotatably orpivotably mounting a pivot rod 34 therein about a vertical axis X₁. Theseparating device 8 can have two spaced securement bands 22 around thedarkened chamber (10-1) with securement fixtures 22 a that secure theseparating device 8 to the pivot rod 34. The pivot rod 34 can have stopmembers 36. The pivot rod 34 can allow the separating device 8 to bepivotably adjusted about axis X₁. The separating device 8 can bepositioned to extend above C-clamp 26 as shown in FIG. 1M, or below asseen in FIG. 1N.

The devices, methods and kits can be used together or separately toobtain of well-separated samples of phytoplankton and/or zooplankton.One of skill in the art will recognize that modifications andadjustments to the device, kits and methods are encompassed by the scopeof the invention described herein.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

EXAMPLES

Devices were developed that provides the conditions necessary toinitiate and direct the movement of zooplankton. Experiments wereconducted in Lake Cochichewick and Willand Pond to evaluate separationefficiency for zooplankton and phytoplankton as measures by zooplanktonbiomass, microcystis equivalents and chlorophyll (a). The evaluationincluded an original design (2013) and an improved design (2014). Therewas no significant difference in zooplankton separation efficiencies foreither lake between sampling years. Significant reduction in amount andvariability of microcystis equivalents and chlorophyll(a) was observedin 2014. The methods and devices allow for the rapid collection ofsamples for surveillance programs and ecological risk assessments. Thisis part of an ongoing study to evaluate the device and method indifferent conditions and over seasonal cycles, and in other environmentswhere questions regarding the presence of toxic substances and theirpotential for bioaccumulation exist.

Example 1

Materials and Procedures

Collection and Processing of Plankton Samples

The study sites included Lake Cochichewick in North Andover, Mass.(August-October 2013) and Willand Pond in Dover, N.H.(September-October, 2013). Lake Cochichewick is classified as amesotrophic system and Willand Pond is classified as an oligotrophicsystem. Concentrated plankton samples for testing were collected fromthe deep sites between the hours of 10 AM-2 PM using a vertical tow bylowering a 50 nm nylon mesh 30 cm open ring conical plankton net fittedwith a 50 um mesh bucket to a depth of 5 m (volume filtered=350 L) andraising vertically at a speed approximating 0.5 m/s. The total number ofplankton samples collected depended upon the number of trials to beconducted that day. For example, if twelve (12) trials are conducted,twelve (12) samples are collected. The concentrated plankton sampleswere placed together in one (1) L. darkened HDPE bottles. Typically,eight (8) to ten (10) concentrated samples are collected in a singlebottle, and two bottles of concentrate collected for testing.

Whole lake water was collected in 1 liter darkened HDPE bottles as asurface grab sample to be used as diluent for the concentrate, and assupply for filtered lake water. The concentrated samples were combinedin a 5 liter container, mixed, and split using a Folsom planktonsplitter until 100 ml aliquots were obtained. The whole lake water(diluent) was combined in a series of 5 liter containers and split usinga Folsom plankton splitter until 850 ml aliquots were obtained. Theindividual concentrate portions (100 mls) were combined with theindividual diluent portions (850 mls) for a total of 950 mls of planktonsample, and placed into darkened HDPE bottles. Typically 24 bottles ofplankton were prepared in this manner. Filtered lake water was preparedby filtering 1 liter of whole lake water through a 50 um mesh ring netand placing it in a 1 liter beaker. Prior to use in the separationdevice, filtered lake water samples were analyzed followingquantification.

Plankton Separation Efficiency:

Step 1. A separation device was suspended using a sling apparatus. Thecollection tube was closed off using the ratchet clamp, and filled withfiltered lake water. The collection tube was then physically separatedfrom the chamber with the use of a black rubber stopper attached to aplastic rod. The plankton sample was then poured into the chamber. Whenvolume series, time series or calibration series were conducted, as manyseparation devices as needed were prepared in this manner concurrently.For example, when a time series for 0, 10, 20 and 30 minutes wasconducted, four (4) separation devices were prepared. The rubber stopperwas removed, the lid placed on top of the chamber and the timer set forthe desired time interval.

Collection of Zooplankton and Phytoplankton

Step 2. At the desired time interval, the desired volume of sample wasreleased from the collection tube by opening the ratchet clamp,dispensing the sample into an appropriate container, and then closingthe ratchet clamp. This sample was marked as the “Z” (zooplankton)portion. The remainder of the sample was released from the collectiontube by opening the ratchet clamp and dispensing the sample into a 1 L.carboy. This sample was marked as the “P” (phytoplankton) portion.

Quantification

Step 3. Phycocyanin (PC) and Chlorophyll (a) (Chla) for the “Z” portionand “P” portion were quantified in vivo, using intact cells withoutfiltration or extraction, using a two-channel hand held AquaFluorfluorometer (Turner Designs, Sunnyvale, Calif.). Using a disposablepipette, 5 mls of each “Z” portion and each “P” portion was placed intoa Turner Design methacrylate cuvette. Large specimens of zooplanktonwere removed from the cuvette using a small tipped disposable pipetteprior to analysis. The filled cuvette was placed in the fluorometer and,using channel A, the relative fluorescence units for PC were recorded.Without removing the cuvette from the instrument, channel B was selectedand relative fluorescence units for Chla were recorded. PC (excitationat 595 run, emission at 670 nm) was standardized (R2=0.99, p<0.0000,Microcystis equivalents (MIC eq.)=1369 (x)+4245) using M. aeruginosa2385. Chlorophyll a (excitation at 460 nm, emission>665 nm) wasstandardized (R2=0.99, p<0.000, Chla=8624 (x)−120812) with solidsecondary standard (No. 8000-952, Turner Designs). The PC and Chla valueof the “Z” portion was adjusted (Adj. Z) to account for the backgroundin the filtered water. The MIC (eq). and Chl(a) concentration/ml wereadjusted to reflect volumes collected. The proportion of MIC (eq.) orChl(a) (separation efficiency) in the “Z” portion for each sample wascalculated as follows:(Adj. Mic. eq. “Z”)I(Adj. Mic. eq. “Z”)+(Mic. eq. “P”)=Separationefficiency for cyanobacteria  Eq. 1(Adj.Chl(a)“Z”)/(Adj. Chl(a)“Z”)+(Chl(a)“P”)=Separation efficiency forphytoplankton  Eq, 2

Step 4. The “Z” portion was preserved using 5% formalin/sucrose. SeeHaney, J. F. & D. J. Hall, 1973, “Sugar coated Daphnia: A preservationtechnique for Cladocera,” Limnol. Oceanogr. 16: 970-977. The “P” portionwas filtered through a 50 um mesh ring net, backwashed and brought to anappropriate volume using filtered lake water, and preserved using 5%formalin/sucrose.

Identification and Counting

Step 5. Zooplankton in each “Z” and “P” sample were identified,enumerated and measured. A minimum of 200 individuals were counted in aknown subsample volume. The body length (and width as needed) of thefirst 20 individuals encountered for each genus and/or species wasmeasured. If needed, the count data of the “P” portion was adjusted(Adj. P) to reflect the proportions of sample removed above to quantifyphycocyanin and Chlorophyll (a). The count data for the “Z” and “P”portion were adjusted to reflect total sample volume. Dry weightestimates for Daphnia spp. Diaphanosoma spp., Copepods and Bosmina spp.were calculated according to Bottrell, H. H., A. Duncan, Z. M. Gliwicz,E. Grygierek, A. Herzig, A Hillbricht-Ilkowska, H. Kurosawa, P. Larsson,and T. Weglenska, 1976, “A review of some problems in zooplanktonproduction studies,” Norw. J. Zool., 24:419-456, Chydorus spp. wascalculated according to Dumont, H. J., I. van de Velde, and Dumont, S.,1975, “The dry weight estimate of biomass in a selection of Cladocera,Copepoda and Rotifera from the plankton, periphyton and benthos ofcontinental waters,” Oecologia, 19:75-97. Rotifers were calculatedaccording to EPA Great Lakes National Program Office, 2003, “Standardoperating procedure for zooplankton analysis,” LG403, Revision 3 Feb.2003, and nauplii were assigned a constant dry weight of 0.40 ug. Theproportion of zooplankton biomass (separation efficiency) in the “Z”portion for each sample was calculated as follows:(Dry wt. “Z”)/(Dry wt. “Z”)+(Dry wt.(Adj.)“P”)=Biomass separationefficiency for zooplankton  Eq. 3

The proportionate values were arcsine transformed (Zar, Jerrold H.,Biostatistical Analysis, Prentice-Hall, Inc. New Jersey, 1974 ed.) andappropriate statistical analysis performed.

Assessment:

The desired volume of the darkened chamber was one (1) liter. Thephysical requirements of the collection tube (transparent, preferablyconical and allowing migrating animals to exceed 48° to the normal)suggested a long, narrow tube or tubing. A tube which measured1″(D)×8″(L), had a maximum volume of 75 mls, and exceeded the 48°angular criteria at a volume of approximately 50 mls was used. In thisembodiment, a stopper was used to provide a temporary physicalseparation between the chamber and the collection tube. The separationtime and volume of the zooplankton and/or phytoplankton samples to becollected were undetermined. Experiments were designed to evaluate thedevice performance for separation of zooplankton with samples from LakeCochichewick (August 1) using the methods described in Steps 1-2 andSteps 4-5.

First Experiment

The first experiment (August 1) was designed to evaluate separationefficiencies for zooplankton using the methods described in Steps 1-2and 4-5.

The results in FIG. 2 indicate that the greatest separation efficiencyoccurred at T=40 minutes for macrozooplankton and microzooplankton.Separation efficiencies greater than 90% occurred at T=20 minutes formacrozooplankton.

Second Experiment

The experimental design was modified to evaluate separation efficienciesusing the methods described in Steps 1-5. Additional experiments wereconducted using samples from Lake Cochichewick (September 4) and WillandPond (September 5) with the results shown in FIG. 3A and FIG. 3B. Themacrozooplankton consistently had the highest mean separation efficiencyfor both lakes. The macrozooplankton comprised 89% of the biomass inLake Cochichewick and 71% of the biomass in Willand Pond.

FIG. 4A and FIG. 4B are plots showing calibration curve formacrozooplankton (solid line), cyanobacteria (dashed line), and allphytoplankton (dotted line) for Lake Cochichewick (A) and Willand Pond(B) on Sep. 4 and Sep. 5, 2013, respectively. Confidence intervals areshown in gray.

FIG. 5A and FIG. 5B compare the September calibration curves with datafrom samples taken in October for Lake Cochichewick (FIG. 5A) andWilland Pond (FIG. 5B). The macrozooplankton comprised 93% of thebiomass in Lake Cochichewick and 91% of the biomass in Willand Pond.

The calibration curves suggest that the researcher could select avariety of times to allow for the separation of the zooplankton and thephytoplankton depending on project objectives. For this study, theobjective was to collect a known volume at a specific time that wouldcontain the greatest biomass of macrozooplankton with the least biomassof cyanobacteria. FIGS. 4A and 4B suggest that, in this context, theresearcher should wait 30 minutes before the collection of a 50 mlzooplankton sample, and then collect the remaining 900 mls for acyanobacterial sample.

Discussion

The results from this study suggest that a rapid method can be used inthe field to separate plankton into the component parts. The datasuggest that similar results may be obtained when the researcher followsthe standard operating procedure and uses a device having designelements sufficient to facilitate a positive phototactic response.

Ambient light was used to simulate the spectral distribution ofirradiance in the natural system. Filtered lake water was used toaddress issues related to dissolved substances and the response of thezooplankton to rapid changes in water temperature.

The design of the separation device with the completely darkened chamberand a transparent collection tube located at a 90 degree verticalposition allows for direct illumination which has an angulardistribution that approximates 0 (zero).

The design of the separation device with a completely darkened chamberand transparent collection tube provides the conditions to initiate apositive phototactic response to a sudden change in light intensity(Buchanan, C. B. Goldberg and R. McCartney 1982, “A laboratory methodfor studying zooplankton swimming behaviors,” Hydrobiologic 94, 77-89).Although measurements of the light intensity were not taken during theseexperiments, it was assumed that the light intensity exceeds thethreshold for instantaneous relative change in light intensity of 0.2 uEm-2 s-1 (the rheobase) necessary for photobehavior to occur. Theorientation of the device (darkened above, light below) serves tofacilitate and reinforce body axis orientation which results from aresponse to the spatial change in light intensity (light/darkboundaries, contrast and shadows). Daphnia magna have been shown toorient their movements away from overhead shadows as a flight responsefrom predators.

The length of the separation device and the volume of the zooplanktonsample to be collected are determined for migration potential andcontrast orientation. By creating a sudden stimulus of dark to light tomaximize migration rates, the maximum migration distance of 42 cm wasachieved within a specified time. Additionally, by leveraging theinfluence of contrast orientation by driving the zooplankton past theangle of 48° to the normal, optimal conditions were achieved. Theoptimum volume to be collected using this collection tube was determinedto be 50 mls.

In addition to facilitating migration and contrast orientation, theincidental collection of phytoplankton in the zooplankton sample and thezooplankton in the phytoplankton was considered. Optimizing theseparation and collection for each portion of the plankton was achieved.Separation efficiency using a method collecting a 50 ml sample versus analternative method collecting a 250 ml sample demonstrated the superiorquality of the samples obtained using the method described herein.

In spite of achieving separation efficiencies that exceeded ourexpectations, it is still necessary to account for the variability weobserved. In regard to the zooplankton, it is possible that thecomposition and distribution of the biomass had an influence on theseparation efficiencies that was observed. It is also possible that laketrophic status exerts a significant influence on separation efficiency.We remain somewhat puzzled at the level of incidental capture of thephytoplankton, specifically the cyanobacteria, in the zooplanktonsamples and the variability of the results. The calibration curvessuggest that after 30 minutes, the levels obtained would be no differentthan levels obtained from a completely mixed sample. It is unknownwhether the incidental capture is from a process of sinking or thecombined influence of entrainment and convectional streaming. Sincecyanobacteria typically contain gas vacuoles, the phenomenon of sinkingdoes not appear to be the answer.

Example 2

In further examples, data is presented from two separation devices.

Prototype #1 Design elements: The device contained design elements asshown in FIG. 1A. The darkened chamber was smooth walled and conical inshape, with a volume of 1 L. The chamber was constructed to preventlight from entering the chamber during the separation phase (tightfitting lid as needed). There was a temporary darkened physicalseparation between the chamber and collection tube with a diameter ofapproximately 21.5 mm. This diameter was large enough to allow for anarrow stimulus beam of light to be refracted at an angle approximating45° (or less) to the vertical plane and to meet the spatial needs of themigrating zooplankton. A stopper was used to provide a temporaryphysical separation between the chamber and the collection tube. Thetransparent collection tube allowed for the maximum amount ofillumination and was conical in shape to facilitate the collection ofzooplankton. The length of the collection tube was such that thezooplankton could migrate past a 45° angle to the vertical plane withinthe tube. A tube was used with maximum diameter (21.5 mm), minimumdiameter (5 mm), length of 150 mm and a maximum volume of 75 mls withrubber tubing (diameter 7 mm) and a clamp. External support to thedevice was provided via external rings and a sling device.

Prototype #2 design elements: The device contained design elements asshown in FIG. 1D. The design was modified to reduce the variability inseparation efficiency and to simplify sample handling. It was assumedthere was potential for leakage around the rubber stopper and mixingduring its removal, thereby increasing the amount of phytoplankton foundin the zooplankton sample. To improve the design, an adapter with aminimum diameter of 20 mm was used as the temporary darkened physicalseparation. This was a diameter sufficient to allow for a narrowstimulus beam of light to be refracted at an angle approximating 45° (orless) to the vertical plane and to meet the spatial needs of themigrating zooplankton. The conical collection tube, rubber tubing andclamp was replaced with a cylindrical collection cartridge (25 mmdiameter) that would continue to meet the spatial needs of the migratingzooplankton and simplify sample handling. Rings were added to provideoptions for external support.

Collection and processing of plankton samples: The two study sitesincluded Lake Cochichewick in North Andover, Mass., USA (42° 19.7′N:71°54.9′W) and Willand Pond in Dover, N.H., USA (43° 43.2′N: 70°29.6′W).Lake Cochichewick is classified as a mesotrophic system and Willand Pondis classified as an oligotrophic system (Carlson, 1977). The deep siteswere accessed by kayak. Concentrated plankton samples were collectedbetween the hours of 10 AM-2 PM using a vertical tow by lowering a 50 umnylon mesh 30 cm open ring conical plankton net fitted with a 50 um meshbucket to a depth of 5 m (total volume filtered=350 L) and raisingvertically at a speed approximating 0.5 m/s. The total number of samplescollected depended upon the number of trials to be conducted that day.For example, if 12 trials were to be conducted, 12 samples would becollected. The concentrated plankton samples were placed together in 1 Ldarkened HDPE (high density polyethylene) bottles. Typically 8-10concentrated samples would be collected in a single bottle, and 2bottles of concentrate collected for testing. Whole lake water wascollected in 1 L darkened HDPE bottles as a surface grab sample to beused as diluent for the concentrate and as a supply for filtered lakewater. The concentrated samples were combined in a 5 L container, mixed,and split using a Folsom plankton splitter until 100 ml aliquots wereobtained. The whole lake water (diluent) was combined in a series of 5 Lcontainers and split using a Folsom plankton splitter until 900 mlaliquots were obtained. The individual concentrate portions (100 mls)were combined with the individual diluent portions (900 mls) for a totalof 1 L of plankton sample, and placed into 1 L darkened HDPE bottles.Typically, 24 bottles of plankton were prepared in this manner. Filteredlake water was prepared by filtering 1 L of whole lake water through a50 um mesh ring net and placing it in a 1 L beaker. Prior to use in theseparation device, filtered lake water samples were analyzed followingStep 3 below.

Plankton Separation-Step 1.

Protoype #1. The separation device was suspended using a slingapparatus. The collection tube was closed off using the ratchet clamp,and filled with filtered lake water. The collection tube was physicallyseparated from the chamber with the use of a black rubber stopperattached to a plastic rod. The plankton sample was poured into thechamber. The rubber stopper was removed, the lid placed on top of thechamber and the timer set for the desired time interval. When volumeseries, time series or calibration series were conducted, as manyseparation devices as needed were prepared in this manner concurrently.For example, when a time series for 0, 10, 20 and 30 minutes wasconducted, 4 separation devices were prepared.

Prototype #2: The collection cartridge was attached to the end of theadapter, filled with filtered lake water and then closed. The planktonsample was placed into the chamber. The adapter/collection tube wasscrewed onto the chamber, which was then suspended with a sling. Theadapter was opened and the timer set for the desired time interval.

Plankton Separation-Step 2.

Prototype #1. At the desired time interval, the desired volume of samplewas released from the collection tube by opening the ratchet clamp,dispensing the sample into a 100 ml sample jar, and then closing theratchet clamp. This sample was marked as the “Z” (zooplankton) portion.The remainder of the sample was released from the collection tube byopening the ratchet clamp and dispensing the sample into a 1 L carboy.This sample was marked as the “P” (phytoplankton) portion

Prototype #2. At the desired time interval, the adapter was closed andthe collection cartridge removed from the bottom of the adapter. Thesample was dispensed into a 100 ml sample jar and marked as the “Z”(zooplankton) portion. The chamber was then inverted and the adapterremoved. This sample was marked as the “P” (phytoplankton) portion.

Plankton Separation-Step 3.

Phycocyanin (PC) and Chlorophyll (a) (Chla) for the “Z” portion and “P”portion were quantified using a two-channel hand held AquaFluorfluorometer (Turner Designs). Using a disposable pipette 5 mls of each“Z” portion and “P” portion was placed into a 5 ml vial, frozen and thenthawed. The thawed sample was placed into a methacrylate cuvette. Thefilled cuvette was placed in the fluorometer and using channel A, therelative fluorescence units for PC were recorded. Without removing thecuvette from the instrument, channel B was selected and relativefluorescence units for Chla were recorded. PC (excitation at 595 nm,emission at 670 nm) was standardized (R²=0.99, p<0.0000, Microcystisequivalents (MIC eq.)=1369 (x)+4245) using M. aeruginosa 2385. Chla(excitation at 460 nm, emission>665 nm) was standardized (R²=0.99,p<0.000, Chla=8624 (x)−120812) with solid secondary standard (No.8000-952, Turner Designs). The PC and Chla value of the “Z” portion wasadjusted (Adj. Z) to account for the background in the filtered water.The MIC eq. and Chla concentrations/ml were adjusted to reflect thevolumes collected. The proportion of MIC eq. or Chla (separationefficiency) in the “Z” portion for each sample was calculated asfollows:Adj. MIC eq. “Z”/Adj. MIC eq. “Z”+MIC eq. “P”=Separation efficiency forcyanobacteria  (1)Adj. Chla “Z”/Adj. Chla “Z”+Chla “P”=Separation efficiency forphytoplankton  (2)

Plankton Separation-Step 4.

The remaining “Z” portion was preserved using 5% formalin/sucrose (Haney& Hall, 1973). The remaining “P” portion was filtered through a 50 ummesh ring net, backwashed with a wash bottle filled with filtered lakewater, brought to an appropriate volume using filtered lake water, andpreserved using 5% formalin/sucrose.

Plankton Separation-Step 5.

Zooplankton in each “Z” and “P” sample were identified, enumerated andmeasured using an Amscope T370B-9M compound microscope, a 9.1 megapixelUSB 2.0 digital camera, Amscope Version 3.7 digital imaging software andan IBM Think pad. A minimum of 200 individuals were counted in a knownsubsample volume. The body length (and width as needed) of the first 20individuals for each genus and/or species was measured. If needed, thecount data of the “P” portion was adjusted (Adj. P) to reflect theproportions of sample removed in Step 3 to quantify phycocyanin andChlorophyll (a). The count data for the “Z” and “P” portion wereadjusted to reflect the total sample volume. Dry weight estimates ofbiomass for cladocerans (Daphnia spp., Diaphanosoma and Bosmina) andcopepods and were calculated according to Bottrell (1976). Dry weightestimates of biomass for the cladoceran Chydorus sphericus. wascalculated according to Dumont (1975). All nauplii were assigned aconstant dry weight of 0.40 ug. Dry weight estimates of biomass forrotifers were calculated according to EPA (2003). Values recordedincluded “Macrozooplankton” and “Microzooplankton”. Zooplankton includedas “Macrozooplankton” considered the findings of Lampert, W. and B. E.Taylor, 1985, “Zooplankton grazing in a eutrophic lake: Implication ofvertical migration,” Ecology 66:68-92, Lampert, W., W. Fleckner, H. Raiand B. E. Taylor, 1986, “Phytoplankton control by grazing zooplankton: Astudy on the spring clear-water phase,” Limnol. Oceanogr. 31(3):478-490, Watras, C. J. and N. Bloom, 1992, “Mercury and methylmercury inindividual zooplankton: Implications for bioaccumulation,” Limnol.Oceanogr., 37(6):1313-1318, and Back, R. C., V. Visman, and C. J.Watras, 1995, “Microhomogenization of individual zooplankton speciesimproves mercury and methylmercury determinations,” Can. J. Fish. Aquat.Sci., 52: 2470-2475 and included any genus and/or species whichcomprised greater than 1.0% of the total biomass of the sample.

The zooplankton biomass separation efficiency for each sample wascalculated as follows:Dry wt. “Z”/Dry wt. “Z”+Dry wt. Adj. “P”=Zooplankton biomass separationefficiency  (3)

All proportionate values were arcsine transformed (Zar, Jerrold H.,“Biostatistical Analysis,” Prentice-Hall, Inc. New Jersey. 1974 ed.).Studentized T-tests and analysis of variance (ANOVA) were conductedusing SigmaPlot V. 12.5.

Assessment

An experiment in Lake Cochichewick (1 Aug. 2013) using prototype #1 wasdesigned to evaluate separation efficiencies for zooplankton using themethods described in Steps 1-2 and Steps 4-5. Separation efficiencycurves as shown in FIG. 2 indicate that the maximum separationefficiency occurred at T=40 minutes for Macrozooplankton (97%) andMicrozooplankton (85%). Separation efficiencies greater than 90%occurred at T=20 minutes for Macrozooplankton (95%).

The experimental design was modified to evaluate separation efficienciesfor zooplankton and phytoplankton using the methods described in Steps1-5. This would allow determination of the optimal separation time thatwould provide samples with the greatest amount of zooplankton biomass(with minimal phytoplankton) and phytoplankton biomass (with minimalzooplankton). Experiments were conducted using samples from LakeCochichewick (4 Sep. 2013, 10 Oct. 2013) and Willand Pond (5 Sep. 2013,16 Oct. 2013). FIGS. 7A and 7B show the separations formacrozooplankton, microcystis equivalents and chlorophyll(a) for LakeCochichewick. The mean values for macrozooplankton ranged between 90-95%(September) and 82-89% (October). The mean values for microcystisequivalents ranged between 3-5% (September) and 1-5% (October), whilethe chlorophyll(a) values ranged from 2-3% (September) and 6-9%(October). The macrozooplankton found in Lake Cochichewick in Septemberand October included Diaphanosoma brachyurum, Diaptomus spp. andMicrocyclops rubellus. FIGS. 7C and 7D show the separations formacrozooplankton, microcystis equivalents and chlorophyll(a) for WillandPond. The mean values for macrozooplankton ranged between 81-89%(September) and 79-84% (October). The mean values for microcystisequivalents ranged between 3-8% (September) and 5-14% (October), whilethe chlorophyll(a) values ranged from 3-11% (September) and 6-17%(October). The macrozooplankton found in Willand Pond in September andOctober included Daphnia ambigua, Daphnia catawba, Diaptomus spp. andMesocyclops edax.

The experiments were repeated for Lake Cochichewick (29 Oct. 2014) andWilland Pond (6 Sep. 2014) using prototype #2. FIGS. 8A, 8B, 8C and 8Doffer a comparison of the 2013 and 2014 experiments. In LakeCochichewick, separation efficiencies for macrozooplankton (78-89%),microcystis equivalents (4-5%) and chlorophyll(a) (4%) were observed. InWilland Pond, separation efficiencies for macrozooplankton (69-83%),microcystis equivalents (4-6%) and chlorophyll(a) (3%) were observed.Two additional macrozooplankton were found in Lake Cochichewick in 2014,including Daphnia ambigua and Daphnia mendotae, while themacrozooplankton found in Willand Pond remained unchanged. Theexperiments confirmed that objectives have met to reduce the variabilityin separation efficiency with an improved design of the device. Analysisof variance revealed that the zooplankton separation efficiencies werenot significantly different from 2013 to 2014 for either lake. In LakeCochichewick, the amount of chlorophyll(a) was significantly reduced atT=20 minutes (p=0.024) and T=30 minutes (p=0.049). In Willand Pond, theamount of microcystis equivalents was significantly reduced at T=30minutes (p=0.018) and the amount of chlorophyll(a) was significantlyreduced at T=20 minutes (p=0.009). The reduction in the variability ofthe data was evidenced by the decrease in the standard deviation for themicrocystic equivalents and chlorophyll(a) values from 2013 to 2014.

FIG. 9 provides evidence as to the importance of the spatial needs ofthe migrating zooplankton. This experiment evaluated the effect of theminimum diameter of the adapter that provided the temporary darkenedphysical separation. The experiments were conducted with ambient andartificial light, as well as adapters with minimum diameters of 20 mmand 13 mm. During the experiments with the 20 mm adapter, it was notedthat the animals migrated freely, appearing in the collection cartridgewithin a minute of opening the ball valve. However, when the 13 mmadapter was used, 2 of the 3 collection cartridges did not have anyzooplankton in them after as many as 5 minutes. The cartridges needed tobe gently tapped to release the animals that were apparently cloggingthe opening. T-tests revealed that there was no significant differencein separation efficiency when using ambient light or artificial (LED)light for either the 20 mm or 13 mm adapter. However, the separationefficiency for the 20 mm adapter was significantly higher than the 13 mmadapter under ambient (t=3.54, df=4, p=0.024) and artificial (t=4.90,df=4, p=0.008) illumination.

The separation efficiencies provide greater insights into thephototactic behavior, under these conditions, of an in-situ zooplanktoncommunity taken from two distinct waterbodies. Although there was someoverlap in the zooplankton community composition between the two lakes(e.g., D. ambigua, Diaptomus spp, copepodites, nauplii), there weredistinct genus and species as well (e.g., D. catawba, D. mendotae,Diaphanosoma brachyurum, Microcyclops rubellus and Mesocyclops edax.).Community composition and distribution could ultimately influence theseparation efficiencies that could be achieved for any given waterbody.Additionally, knowledge of the separation efficiencies of the individualzooplankters could potentially allow for the selection of a separationtime based upon a target organism. FIG. 10 provides a summary of theindividual zooplankter behavior that was observed in samples taken fromLake Cochichewick and Willand Pond in 2014. These are comparable tothose previously observed as shown in Table 1 of FIG. 11 and Table 2 ofFIG. 12, respectively.

The design of the separation device with a completely darkened chamberand transparent collection tube provides the conditions necessary toinitiate a positive phototactic response to a sudden change in lightintensity (Buchanan, C. B. Goldberg and R. McCartney, 1982, “Alaboratory method for studying zooplankton swimming behaviors,”Hydrobiologic, 94, 77-89). Although the light intensity was not measuredduring these experiments, it was assumed that the light intensityexceeded the threshold for instantaneous relative change in lightintensity of 0.2 uE m-2 s-1 (the rheobase) necessary for photobehaviorto occur (Ringelberg, J., 1964, “The positively phototactic reaction ofDaphnia magna Straus: a contribution to the understanding of diurnalvertical migration,” Neth. J. Sea Res., 2:319-406, Daan, N. and J.Ringelberg, 1969, “Further studies on the positive and negativephototactic reaction of Daphnia magna Straus,” Neth. J. Zool.,19:525-540). A positive phototactic response could be anticipated as aresult of exposure to a narrow stimulus beam (Forward, R. B. Jr., 1988,“Diel vertical migration: Zooplankton photobiology and behavior,”Oceanogr. Mar. Biol. Annu. Rev., 26: 361-393) (highly directional light)with an angular light distribution that approximates 0° (Schallek, W.,1942, “The vertical migration of the copepod Acartia tonsa undercontrolled illumination,” Biological Bulletin, 84:98-106). Body axisorientation would result from dorsal beam contrast (45° or less)(Ringelberg, J., 1964, “The positively phototactic reaction of Daphniamagna Straus: a contribution to the understanding of diurnal verticalmigration,” Neth. J. Sea Res. 2:319-406) (Ringelberg, J., B. J. G. Flikand R. C. Buis, 1975, “Contrast orientation in Daphnia magna and itssignificance for vertical plane orientation in the pelagic biotope ingeneral,” Neth. J. Zool., 25:454-475) that would control the directionof movement in the vertical plane. The orientation of the device(darkened above, light below) serves to reinforce body axis orientationas a flight response from predators.

An adapter with a minimum diameter of 20 mm was used as the temporarydarkened physical separation. This was a diameter sufficient to allowfor a narrow stimulus beam of light to be refracted at an angleapproximating 45° (or less) to the vertical plane and to meet thespatial needs of the migrating zooplankton. A cylindrical collectiontube (25 mm diameter) continuously assured that the spatial needs of themigrating zooplankton would be met. Although experiments were notconducted on the behavior of the migrating zooplankton in devices withother elements (i.e., rubber tubing) with small diameter (e.g., 13 mm orless), it is assumed that the response would be similar. Consequently,it is possible that elements of a separation device with diameters lessthan 13 mm may inhibit the movement of migrating zooplankton.

The volume of the zooplankton sample to be collected needed to considermigration potential and contrast orientation. By creating a suddenstimulus of dark to light to maximize migration rates, (Buchanan,Goldberg and McCartney 1982) it could be ensured that the maximummigration distance of 42 cms could be achieved within a specified time(Daan & Ringelberg 1969). Additionally, it was needed to leverage theinfluence of contrast orientation by driving the zooplankton past theangle of 48° to the normal. The optimum volume to be collected using acollection tube was determined to be 50 mls.

In regards to procedural issues, favorable conditions were establishedfor the response to occur by using ambient light to simulate thespectral distribution of irradiance in the natural system. Filtered lakewater was used to address issues related to dissolved substances andresponse of the zooplankton to rapid changes in water temperature.(Buchanan, Goldberg and McCartney, 1982).

The device is easily assembled and can be used to obtain well separatedin situ samples of phytoplankton and zooplankton. The samples can easilybe processed on site, thereby reducing valuable time either in the fieldor in the laboratory. Issues related to sample handling and transportwere considered, and how that might affect the design of the collectiontubes. Collection tubes containing samples of live zooplankton can besealed with a cap and easily transported. The phytoplankton can beeasily transported by placing a cap on the darkened chamber. To simplifytransport and reduce processing time in the lab, dried zooplankton andphytoplankton samples can be obtained while in the field. This wouldalso reduce the possibility of bacterial contaminations of the samples.Filter cones and modified collection tubes were developed to allow forthe discharge of water. The filter cones can be placed in dryingchambers for 2-8 hours and then placed into desiccators. There arecompeting limitations to the device and the method that relate to avacuum being created within the chamber and incremental clogging of thefilter cone as the phytoplankton sample is being discharged. Theselimitation can be overcome by the sizing of the filter cones andincluding agitation ports in the collection tubes.

Discussion:

The experiments described herein provide novel data, using an in situsample to quantify phototactic behavior under a controlled setting fromtwo distinctly different water bodies. Although phototaxis has beenpreviously used to separate plankton, there are no published studiesthat describe the actual separation efficiencies that could be achieved.From visual observations, phototactic behavior can be used to harvestzooplankton. The composition of the sample was unknown, however it wasassumed that macrozooplankton would migrate more quickly thanmicrozooplankton. Additionally, the amount of incidental capture of thephytoplankton portion was completely unknown. The harvest achieved forthe zooplankton portion surpassed expectations and provided insight intothe level of incidental phytoplankton capture that could be anticipated.Conversely, a phytoplankton portion can be harvested that would berelatively free of zooplankton biomass. The passive nature of the deviceproved to be of great value, as other tasks could be conducted while thesample was separating, thereby saving valuable time.

In regards to the zooplankton, it is possible that the composition anddistribution of the biomass had an influence on the separationefficiencies that were observed. It is also possible that lake trophicstatus exerts a significant influence on separation efficiency. Thelevel (5% or less) of incidental capture of the phytoplankton ispuzzling, specifically the cyanobacteria, in the zooplankton samples. Itis assumed that what is observed as incidental capture is a result ofdepuration as the zooplankton move from an environment of highconcentration of phytoplankton to a lower concentration.

It is anticipated that samples obtained after using the method anddevice would yield relatively precise measures of biomass and weightspecific toxicity for zooplankton and phytoplankton. The phytoplanktoninformation could be used to provide a profile of exposure potentialacross a range of waterbodies and to support decisions regarding useattainability. The zooplankton information could be used to quantifytransfer between the two trophic levels and provide insight into thepotential for further bioaccumulation.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A plankton separating device comprising: adarkened chamber having an outer perimeter surrounding a verticalcentral axis, and a maximum outer perimeter dimension that extendsdownwardly to a darkened narrowed section which ends at a port at thebottom, wherein the darkened narrowed section has an inner openinghaving a size above 13 mm and up to 21.5 mm ±2 mm and an openableclosure; and a light permeable collection tube extending along thevertical central axis attached to the port for allowing highlydirectional ambient light to enter the collection tube and pass upwardlythrough the darkened narrowed section and inner opening which areconfigured to produce a narrow defined beam of light upwardly into thedarkened chamber along the vertical central axis without inhibitingzooplankton migration therethrough when the closure is open, wherein thecollection tube is of sufficient length to reinforce migration of thezooplankton downwardly, by extending downwardly away from the darkenedchamber beyond a point that makes about a 20°±2° angle to the verticalcentral axis while extending to a nearest location of maximum outerperimeter dimension of the darkened chamber, thereby providing acontrast shadow from the darkened chamber relative to the collectiontube simulating a predator to zooplankton, and a length in thecollection tube for zooplankton to migrate from and move away from thedarkened chamber a sufficient distance to minimize zooplankton collectedin the collection tube from migrating back into the darkened chamber,thereby separating plankton into its component parts, wherein theclosure is configured to be changed from a closed state to an openstate.
 2. The device of claim 1, wherein the closure is a stopper orvalve.
 3. The device of claim 1, wherein the darkened chamber isconfigured to be positioned above the collection tube, the collectiontube being elongated and extending from the darkened chamber along thevertical central axis, starting beyond a point that makes about a 48°angle to the vertical central axis while extending to the nearestlocation of maximum outer perimeter dimension of the darkened chamber.4. The device of claim 1, wherein the collection tube is transparent. 5.The device of claim 1, wherein the collection tube has one of tapered orstraight side walls.
 6. The device of claim 1, in which the outerperimeter of the darkened chamber and the side walls of the collectiontube are generally round.
 7. The device of claim 1 in which at leastabout 40% of length of the transparent collection tube extends beyondsaid point that makes said about a 20° angle.
 8. The device of claim 4in which the darkened chamber and the collection tube have outerdiameters with a darkened chamber OD_(b) to transparent collection tubeOD_(t) ratio of about 3-3.5 to 1, the transparent collection tube havinga length with a transparent collection tube length to OD_(t) ratio ofabout 3.9-5.2 to
 1. 9. The device of claim 1 in which the darkenedchamber has a capacity of at least about one liter, and the collectiontube has a capacity of at least about 50 ml.
 10. The device of claim 1in which the inner opening is between the darkened chamber and thecollection tube and size is in the range of 19 to 22 mm across.
 11. Thedevice of claim 4 in which the transparent collection tube has an innerdiameter, at least a portion of which being about 20 mm to about 26 mm.12. The device of claim 1 in which the length of the collection tube isat least about 110 mm.
 13. A plankton separation device comprising: adarkened chamber having an outer perimeter surrounding a verticalcentral axis, and a maximum outer perimeter dimension that extendsdownwardly to a darkened narrowed section which ends at a port at thebottom, wherein the darkened narrowed section has an inner openinghaving a size above 13 mm and up to 21.5 mm±2 mm; and a light permeablecollection tube extending along the vertical central axis and extendingfrom the port for allowing light to enter the collection tube and passupwardly through the darkened narrowed section and inner opening, whichare configured to produce a narrow defined beam of light into thedarkened chamber along the vertical central axis without inhibitingzooplankton migration therethrough, the collection tube of sufficientlength to reinforce migration of zooplankton downwardly, by extendingdownwardly away from the darkened chamber beyond a point that makesabout a 20°±2° angle to the vertical central axis while extending to anearest location of maximum outer perimeter dimension of the darkenedchamber, thereby providing a contrast shadow relative to the collectiontube simulating a predator to zooplankton, and sufficient length in thecollection tube for zooplankton to migrate from and move away from thedarkened chamber a sufficient distance to minimize zooplankton collectedin the collection tube from migrating back into the darkened chamber.